A lab-on-a-chip is a chip where various test processes are performed in a laboratory, for example, separation, refinement, mixing, labeling, analysis, and washing, etc. of a sample, on a chip having a small size. Techniques related to microfluidics and a micro-LHS are typically used in designing the lab-on-a-chip. Also, in manufacturing a chip-structure implementing microfluidics and the micro-LHS, a chip, in which a microchannel is formed at the interior of the chip by using a semiconductor circuit designing technique, has been put on the market. For example, in a microarray chip, material to be detected and a probe to be reacted, e.g. protein or DNA, are adhered on a substrate with a predetermined interval, and material to be coupled with them is detected. Therefore, the microarray chip is used in diagnosing trouble or disease. A chip where proteins are arranged is a microarray protein-chip, and a chip where DNA is arranged is a micro DNA chip. Analytes existing in blood, for example, proteins, antibodies, etc. exist in a very small amount. Therefore, an attempt to increase detecting efficiency by increasing the degree of integration of probes arrayed on a substrate has been continuously performed. As a result, a nanoarray chip, which increases integration of probes to a nano-level, has been reported.
In general, analysis of a sample fluid is typically used in analyzing blood and body fluid collected from a patient and in diagnosing disease through the analysis, as well as in a chemistry and bio-technical field. As such, detecting and analyzing analytes in a very small amount, which are included in sample fluid, such as blood, a body fluid, urine, etc., includes analyzing if a sample fluid reacts against proteins, such as antigens, antibodies, etc., DNA/RNA, a receptor, or other material, which have previously immobilized on a chip, while moving through a channel having a pipe-shaped structure formed in the interior of the chip, through a detecting signal using an optical means, such as a fluorescent material, etc. or an electric means. In analyzing a sample fluid, it is very important in a biochip, as well as a lab-on-a-chip, to analyze a sample in a very minimum amount.
In general, a lab-on-a-chip for analyzing a sample fluid includes: a sample injecting part for supplying sample fluid to the chip; a filtering zone using a paper filter so as to remove noise material, except for analytes in fluids; a reaction zone where binding between anlaytes and detection signal generating material, e.g. fluorescent material is performed, and reaction between the analytes bonded to the detection signal generating material and material, which is immobilized on a substrate to specially react against the analytes, is performed; and a detection zone for detecting anlaytes, such as blood, urine, proteins in body fluids, etc., by using a chip, and detecting detection signals through a connected detection apparatus.
Whole blood includes plasma (46˜63%) and blood corpuscles (37˜54%) having red blood cells (donut-shape, diameter: 7˜8 μm/thickness: 2 μm, 50% of blood), leukocytes (a non-regular spherical shape, diameter: 10˜20 μm), and platelets (diameter: 1˜3 μm). In addition to plasma and blood corpuscle, various kinds of analytes, which can be used in diagnosing troubles and diseases, e.g. protein, antigens and antibodies, ligands, receptors, etc. exist in blood. Therefore, various methods and apparatuses for detecting analytes related to troubles and disease from whole blood has been developed. Meanwhile, blood corpuscles in an extremely small amount, transitional epithelial cell (diameter: 20˜40 μm), and squamous epithelial cell (diameter: 40˜60 μm) are included in urine.
Research for rapidly obtaining various pieces of information from end blood collected in a clinic by using a lab-on-μ chip in analysis of blood has been recently progressed. As a result, a rapid-chip or a rapid-kit has been developed. As shown in FIG. 1, a kit disclosed in U.S. Pat. No. 6,485,982 applied by Armkel, LLC Corp includes a filtering process, in which a blood sample is diluted and a porous membrane is used, as a essential process. In a reaction zone, conjugation between an analyte and a marker so as to allow the analytee to be labeled by the marker, e.g. fluorescent material, bonding between the marker and a probe, and cleaning are performed in the membrane. This kit provides a relatively high effective volume ratio due to low hematocrit since this kit uses a diluted blood sample. However, there is a problem in that the kit is not suitable for quantitative analysis, and pre-processes, such as dilution, etc. make it difficult to rapidly detect analytes (see Table 1).
TABLE 1Volume (μl)Qualitative/Semi-Characteristicquantitative analysis1. Sampling>1002. Filtering183. Labeling (conjugation)84. Binding reaction10.25. Cleaning23.8Effective volume ratio<42%(3 + 4 + 5)/(1)
Filtering (2) is performed in a preprocessing unit in a chip, and labeling (3), binding reaction (4), and cleansing (5) are performed in the reaction zone.
Meanwhile, U.S. Pat. No. 6,905,882 applied by Biosite corp. discloses a chip, which has a structure where flux of fluids can be controlled through a micochannel and material of a substrate (hydrophile property/hydrophobic property), and can be coupled with a known porous membrane so as to be used. That is, it is suggested that the movement speed of fluids in a sample injecting part and the reaction zone can be controlled by selectively using a microchannel or material of a substrate (hydrophile property/hydrophobic property). Also, a chip invented by Biosite corp. has a reaction zone implemented in a fluidic channel instead of a membrane. Therefore, all of conjugation between makers and analytes, reaction between analytes and probes, and cleansing are performed within the microchannel (see table 2). In a case where this chip is used in blood analysis, quantitative analysis is possible, and whole blood can be used without pre-processes, such as diluting, etc. However, due to high hematocrit of a sample, there is a very low effective volume ratio, which means an amount of a sample taking part in reaction, which is required for detecting an initial injected amount of a sample (see table 2). Also, in a case where filtering isn't performed, a large amount of noise materials in addition to analytes moves through a channel so that it is very difficult to obtain an accurate analysis result. Therefore, in order to perform accurate analysis, an additional filtering means or step is necessarily required.
TABLE 2Volume (μl)Characteristicquantitative analysis1. Sampling140~1502. Filtering1053. Labeling (conjugation)14. Binding reaction55. Cleaning25Effective volume ratio  12~22%(3 + 4 + 5)/(1)
Filtering (2) is performed in a preprocessing unit in a chip, and labeling (3), binding reaction (4), and cleansing (5) are performed in the reaction zone.
Other blood analysis chips (a rapid-chip) substantially include a filtering process as an essential process. Filtering, and particularly filtering using a paper filter, causes dead volume of a sample fluid because the sample fluid is absorbed in the filter. Consequently, in order to detect and analyze analytes existing in the sample, a minimum injection amount of a sample (in a case of hematologic analysis, >100 μl) is required. This makes it impossible to detect and analyze analytes existing in fluids in the minimum amount. Thus, research about micro fluidics has been continuously progressed so as to replace a paper filter. However, in reality, dead volume of a sample fluid hasn't been decreased until now.
Thus, the present inventors have developed a module where a filtering zone and a reaction zone for detecting and analyzing analytes in fluids are implemented in a microchannel, and have manufactured a chip having the module. Also, detecting analytes from the minimum amount of sample fluid has been performed. As a result, dead volume of the sample fluid was remarkably reduced so that it was confirmed that quantitative and qualitative analysis can be rapidly performed by using the minimum amount of the sample. Accordingly, based on the above conformed fact, the present invention was invented.